Compositions and methods for treating neoplasia

ABSTRACT

The invention provides therapeutic methods featuring the use of chimeric human/mouse antibodies for the treatment of neoplasia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of international patentapplication No. PCT/EP2011/066791, filed Sep. 27, 2011, that waspublished in English on Apr. 5, 2012 as publication No. WO 2012/041863A1, and claims the benefit of U.S. Provisional Application No.61/386,764, filed Sep. 27, 2010. The entire contents of theaforementioned patent applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 27, 2013, isnamed 313200.00013_SL.txt and is 14,948 bytes in size.

BACKGROUND OF THE INVENTION

Monoclonal antibody (mAb) 4C5 is a murine antibody that specificallyrecognizes both the a and to a lesser extent the β isoforms of the heatshock protein 90 (HSP90). Recently, HSP90 has become a very attractivedrug-target for cancer therapy because most of its client proteins areconsidered to be key molecules in the acquisition of the malignantphenotype. Moreover, emerging data demonstrating the presence of thismolecular chaperone at the surface of cancer cells suggest awide-ranging phenomenon of extracellular chaperoning implicated incancer cell invasion and metastasis.

MAb 4C5 was initially shown to inhibit cell migration processes in vitroduring development of the nervous system by affecting actin cytoskeletalrearrangement and the formation of motile structures, such aslamellipodia. mAb 4C5 selectively binds to the surface pool of HSP90,and significantly reduces melanoma cell invasion and metastasis.Furthermore, mAb 4C5 was shown to inhibit the extracellular interactionbetween HSP90 and the growth factor receptor HER-2 in MDAMB453 breastcancer cells, leading to impaired downstream signalling and reducedcancer cell motility and invasion. Finally, mAb 4C5 was shown to inhibita functional interaction between secreted HSP90 and pro-MMP2 andpro-MMP9, necessary for the activation of these enzymes which isessential for ECM degradation and cancer cell invasion andextravasation.

These combined data suggested that the capacity of mAb 4C5 tospecifically inhibit the extracellular pool of HSP90 without affectingthe wide range of important intracellular roles of this molecularchaperone could have clinical benefits in the treatment of humanmalignancies. However, murine mAbs do not constitute ideal therapeuticagents. An obvious problem with the use of murine mAbs in human clinicaltrials is the potential for the generation of human anti-mouse antibodyresponses. Initial attempts to use murine-derived mAbs in humantherapeutics were hampered because murine antibodies were recognized bya human anti-murine-antibody immune response (HAMA) and the patient'simmune system cut short the therapeutic window. These obstacles havebeen overcome by the advent of recombinant DNA technologies, which haveled to the development of chimeric or humanized antibodies.

SUMMARY OF THE INVENTION

As described below, the present invention provides therapeuticcompositions and methods for the treatment of neoplasia. In particularembodiments, the present invention provides chimeric human/mouseantibodies for use in the treatment of neoplasia. Because suchantibodies are largely human in composition they have a reduced capacityto induce an immune response in a human subject, relative toconventional murine antibodies.

In one aspect, the invention provides an isolated chimeric antibodycomprising a murine kappa L-chain that lacks heavy chains, where themurine immunoglobulin κ light chain constant domain (C_(K)) is replacedwith the corresponding human C_(K) domain or a fragment thereof, wherethe antibody specifically binds HSP90 and is capable of reducing thegrowth and/or the invasiveness of a neoplastic cell. In one embodiment,the murine kappa L-chain has at least 75%, 85%, 90% or 95% identity toSEQ ID NO: 2. In another embodiment, the murine kappa L-chain containsSEQ ID NO: 2. In another embodiment, the murine kappa L-chain containsone or more murine complementarity determining region. In anotherembodiment, the murine complementarity determining region has at least75%) identity to SEQ ID NO: 5, 6, or 7. In another embodiment, themurine light chain contains one or more complementarity determiningregions (CDRs) that is SEQ ID NO: 5, 6, and/or 7. In another embodiment,the murine light chain contains complementarity determining regions(CDRs) SEQ ID NO: 5, 6, and 7. In another embodiment, the antibody hasat least 75%, 85%, 95% or more amino acid sequence identity to SEQ IDNO:4.

In another aspect, the invention features an isolated chimeric antibodycontaining or consisting essentially of SEQ ID NO: 4. In one embodiment,the antibody is a humanized antibody. In another embodiment, theantibody is a monomer, a dimer, or a multimer. In another embodiment,the antibody is isolated from a culture of prokaryotic or eukaryoticcells. In various embodiments of the above aspects, the chimericantibody has a reduced capacity to induce an immune response in a humansubject, relative to a conventional murine antibody.

In still other embodiments of the above aspects, the ability of theantibody or a fragment thereof to reduce growth and invasiveness isassayed using a cancer cell clonogenic assay, a wound healing assay, alung metastatic deposit formation assay, a lung metastasis inhibitionassay, a breast cancer primary tumor growth inhibition assay, bydetecting actin rearrangement, lamellipodia development or anothermorphological marker of invasiveness, by detecting inhibition ofmetastatic lung deposits, by detecting inhibition of lung metastasis, bydetecting delay of primary growth tumors implanted orthotopically inmouse fat pads, or by detecting another marker of efficacy,respectively.

In another aspect, the invention features an isolated polypeptidecontaining the sequence of SEQ ID NO: 4.

In another aspect, the invention features an isolated polynucleotideencoding the chimeric antibody of any previous aspect.

In another aspect, the invention features an isolated polynucleotideencoding the chimeric antibody of a previous aspect or the isolatedpolypeptide of the previous aspect. In one embodiment, thepolynucleotide has the sequence of SEQ ID NO:3.

In another aspect, the invention features an expression vectorcontaining the polynucleotide of any previous aspect positioned forexpression in a cell.

In another aspect, the invention features a cell (e.g., a prokaryotic oreukaryotic cell) containing that expression vector.

In another aspect, the invention features a method for producing thechimeric antibody of the invention, the method containing culturing acell containing an expression vector of the invention under conditionssuitable for expression of the chimeric antibody, and isolating thechimeric antibody from the cultured cell.

In another aspect, the invention features a method of reducing thegrowth and/or invasiveness of a neoplastic cell, the method involvingcontacting a neoplastic cell with a chimeric antibody of any previousaspect or otherwise delineated herein, thereby reducing the growthand/or invasiveness of the neoplastic cell.

In another aspect, the invention features a method of treating a subjecthaving a neoplasia, the method involving administering to a subject atherapeutically effective amount of the chimeric antibody of anyprevious aspect or otherwise delineated herein, thereby treating thesubject.

In another aspect, the invention features a method of treating orpreventing tumor progression or metastasis in a subject having aneoplasia, the method involving administering to a subject atherapeutically effective amount of the chimeric antibody of anyprevious aspect or otherwise delineated herein, thereby treating orpreventing tumor progression or metastasis in the subject. In oneembodiment, the neoplastic cell is a cancer cell or is present in atumor. In another embodiment, the cancer is breast cancer, melanoma,glioblastomas, colon cancer, non-small cell lung cancer or lymphoma. Inanother embodiment, the chimeric antibody is administered systemicallyor locally. In another embodiment, the chimeric antibody is a monomer, adimer or a multimer. In another embodiment, the chimeric antibody iscovalently linked to a functional moiety. In another embodiment, thefunctional moiety is radioactive or is a chemotherapeutic agent.

In another embodiment, the method further involves administering to asubject a therapeutically effective amount of one or morechemotherapeutics. In various embodiments of the above aspects, one ormore chemotherapeutics is selected from any one or more of thefollowing: abiraterone acetate, altretamine, anhydro vinblastine,auristatin, bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide(SEQ ID NO: 8), cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin- caleukoblastine, docetaxol,doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU),cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide,hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine,lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard),melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin,mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone,paclitaxel, prednimustine, procarbazine, RPRI 09881, stramustinephosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine,vincristine, vindesine sulfate, and vinflunine.

In various embodiments of the above aspects, the chimeric antibody has areduced capacity to induce an immune response in a human subject,relative to a conventional murine antibody.

In another aspect, the invention features a pharmaceutical compositionfor the treatment of neoplasia comprising a therapeutically effectiveamount of the chimeric antibody of any previous aspect or otherwisedelineated herein. In another aspect, the invention features apharmaceutical composition for the treatment of neoplasia comprising atherapeutically effective amount of an isolated polypeptide comprisingSEQ ID NO: 4.

In another aspect, the invention features a pharmaceutical compositionfor the treatment of neoplasia comprising a therapeutically effectiveamount of an isolated polypeptide comprising one or more complementaritydetermining regions selected from SEQ ID NO: 5, SEQ ID NO: 6, and SEQ IDNO: 7.

In another aspect, the invention features a kit for the treatment of aneoplasia, the kit comprising a therapeutically effective amount of thechimeric antibody of any previous aspect or otherwise delineated hereinor an isolated polypeptide comprising a sequence selected from SEQ IDSEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 8, SEQID NO: 8, and SEQ ID NO: 7; and directions for using the kit in a methodof any previous aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and 1B show that mAb 4C5 is a kappa L-chain dimer that lacksheavy chains. FIG. 1 A is a western blot of mAb 4C5 run under reducingand non-reducing conditions and probed with antibodies specific for themouse kappa chain, mouse Fab, and Fcγ. Purified mAb 4C5 isolated fromhybridoma cultures was subjected to SDS-PAGE electrophoresis underreducing and non-reducing conditions, followed by immunoblotting with ananti-mouse kappa chain, an anti-mouse Fab, and an anti-Fcγantibody.Under reducing electrophoresis followed by western blot with theanti-Fab antibody, a single 25 kDa-immunoreactive band was observedwhich is identical to the band corresponding to the kappa L-chain asshown by western blot with the anti-mouse kappa chain antibody.Under-non-reducing SDS-PAGE followed by western blot with both theanti-kappa and the anti-Fab antibodies, mAb 4C5 was shown to migrate atapproximately 50 kDa, and not at 150 kDa as a conventional IgG1molecule. No mAb 4C5 immunoreactivity was detected after electrophoresisunder both reducing and non-reducing conditions followed by western blotwith an anti-Fcγantibody. FIG. 1B is a Northern blot of 4C5 hybridomaRNA hybridized with a heavy chain probe. No radioactivity was detectedafter Northern blot analysis of 4C5 hybridoma-derived RNA with a heavychain radiolabelled probe. 2D10- and NSO-derived RNA served as positiveand negative control, respectively.

FIG. 2 shows the nucleotide (SEQ ID NO: 22) and deduced amino acid (SEQID NO: 23) sequences of mAb 4C5. The complementarity determining regions(CDRs) are underlined.

FIGS. 3 A and 3B show that both the mouse-human chimeric 4C5 andrecombinant mouse 4C5 exhibit an electrophoretic mobility that issimilar to that of the parental mAb 4C5. FIG. 3 A is a Coomasie stainedSDS-PAGE of purified antibodies under reducing conditions. SDS-PAGEelectrophoresis of purified antibodies under reducing conditionsfollowed by Coomasie Brilliant Blue-R staining revealed in all cases, anapproximately 25 kDa band corresponding to the L-chain. FIG. 3B is aCoomasie stained SDS-PAGE of purified antibodies under non-reducingconditions. Under non-reducing conditions the antibodies are shown tomigrate as a L-chain dimer. rec 4C5: recombinant 4C5, ch 4C5: chimeric4C5. FIG. 3C shows that monoclonal antibody ch4C5ΔCys is a monomerbecause it migrates around its predicted molecular weight ofapproximately 25 kDa in both reducing and non-reducingconditions_([PC1]).

FIGS. 4A, 4B, and 4C show that recombinant and chimeric 4C5 specificallyrecognize HSP90. FIG. 4A is a western blot of MDAMB453 lysates probedwith a commercially available polyclonal anti-HSP90α antibody, mAb 4C5,recombinant and chimeric 4C5. In all cases a single 90 kDaimmunoreactive band corresponding to HSP90 was observed. FIG. 4B is animmunoblot of anti-HSP90 immunoprecipitants of MDAMB453 lysates probedwith mAb 4C5, recombinant 4C5, and chimeric 4C5. MDAMB453 cell lysateswere immunoprecipitated with anti-HSP90 and immunoblotted with mAb 4C5,recombinant 4C5, and chimeric 4C5. In all cases a single immunoreactiveband was observed. FIG. 4C is an immunoblot of mAb 4C5, recombinant 4C5,and chimeric 4C5 immunoprecipitants of MDAMB453 lysates probed withanti-HSP90α. Reverse immunoprecipitation experiments in MDAMB453 celllysates were performed using mAb 4C5, rec 4C5 and ch 4C5, followed byWestern blot with anti-HSP90α. In all cases a single immunoreactive bandwas observed indicating that both recombinant and chimeric 4C5 retainmAb 4C5 specificity and recognize HSP90.

FIGS. 5 A, 5B, 5C, and 5D show that recombinant and chimeric 4C5 bind tothe cell surface and are not internalized by MDAMB453 cells. FIG. 5A isa photomicrograph of immunofluorescence staining of MDAMB453 cells usingrecombinant and chimeric 4C5. The punctuate immunolabeling indicates thesurface pool of HSP90. Similar results were obtained using mAb 4C5 andanti-HSP90α. Negative controls were performed using an antibody againstthe intracellular protein βtubulin. (not shown) Scale bar: 20 μm. FIG.5B is a photomicrograph of recombinant and chimeric 4C5 binding tointracellular HSP90. Similarly to the murine antibody the recombinantand chimeric 4C5 also recognize the intracellular pool of HSP90following cell permeabilization. Immunofluoresence detection ofintracellular HSP90 in MDAMB453 cells was performed under fixedconditions with 4% paraformaldeyde and permeabilization with 0.1% TritonX-100. Scale bar: 20 μm. FIG. 5C is a photomicrograph of detection ofantibody internalization. Cells were incubated at 37° C. with theantibodies for various time intervals, fixed, and permeabilized. Bindingof the antibodies was visualized using a fluorescence-conjugatedantibody. No internalization of the mAb 4C5, recombinant 4C5, andchimeric 4C5 antibodies was observed—even after 24 of incubation. Incontrast, anti-HSP90α antibody was detected intracellularly after 8hours of incubation. Scale bar: 20 μm. FIG. 5D is a western blot of celllysates derived from MDAMB453 cells treated with mAb 4C5, recombinant4C5, or chimeric 4C5 probed with antibodies against ErbB2, Akt, cRaf,and HSP90. Actin served as a loading control. Treatment with the three4C5 antibodies did not affect the levels of the intracellular kinasestested as compared to controls.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, and 6G show that both recombinant andchimeric 4C5 retain the function blocking properties of mAb 4C5 andinhibit MDAMB453 breast cancer and B16F10 melanoma cell invasion. FIG. 6A is a panel of photomicrographs of the results of an in vitro woundhealing assay. Photographs represent phase-contrast images obtained atzero time (left panel) and at 48 hours (right panel) after scratchformation, showing MDAMB453 cell migration either in control cultures orcultures including anti-HSP90α, mAb 4C5, recombinant 4C5, or chimeric4C5. Scale bar: 200 μm. FIG. 6B is a graphical presentation of the woundhealing assay results. Addition of 200 μg/ml of anti-HSP90α and mAb 4C5in the culture medium resulted in a 48.57% and 55.3% inhibition of woundclosure, respectively when compared to control cultures that wereconsidered as resulting in 100% wound closure. Addition of 200 μg/mlrecombinant 4C5 or chimeric 4C5 in the culture medium resulted in a46.51% and 51.19% inhibition of wound closure, respectively. The barsrepresent the average of three independent experiments±SEM. Within asingle experiment, each condition was tested in triplicate. Statisticalsignificance of differences was tested by Student's T test. The presenceof anti-HSP90α, mAb 4C5, recombinant 4C5, and chimeric 4C5 had astatistically significant effect on the wound closure (p<0.01, p<0.01,p<0.01 and p<0.01 respectively). FIG. 6C is a photomicrograph ofphase-contrast images obtained at time zero (left panel) and at 24 hoursafter scratch formation (right panel), showing B 16 F10 melanoma cellinvasion in a wound healing assay in the presence of 200 μg/ml ofchimeric 4C5. Scale bar: 200 μm. FIG. 6D is a graphical presentation ofthe effect of increasing concentrations of chimeric 4C5 on the closureof the wound. Presence of 50 μg/ml chimeric 4C5 resulted in 16%inhibition of invasion, while addition of 100 μg/ml and 200 μg/mlchimeric 4C5 resulted in 27.9% and 52.3% inhibition of migration,respectively when compared to control cultures that were considered asresulting in 100% wound closure. FIG. 6E is a photomicrograph of deadcells visualized using Trypan Blue dye. Control, mAb 4C5-, anti-HSP90α-,and chimeric 4C5-treated cells were incubated with trypan blue in orderto observe the rate of cell death in each case. Scale bar, 30 μm. FIG.6F is a photomicrograph of control and chimeric 4C5 treated cells afterbeing fixed, permeabilized, and stained with fluorescently labelledphalloidin. Scale bar 40 μm. FIG. 6G is a higher magnification showingphalloidin staining (F-actin). Chimeric 4C5 effectively blocks spreadingof lamellipodia. Scale bar: 16 μm. FIG. 6H shows that ch4C5ΔCys inhibitsB16F10 mouse melanoma cell migration in an in vitro wound healing assay.Photographs represent phase-contrast images obtained at zero time (leftpanel) and at 24 hours (right panel) after scratch formation, showingB16F10 mouse melanoma cell migration either in control cultures orcultures including ch4C5ΔCys_([PC2]).

FIGS. 7 A and 7B show that chimeric 4C5 significantly reduced the numberof cells in each individual colony formed by MDAMB231 human breastcancer cells in a clonogenic assay. FIG. 7A is a photomicrograph of theresults of the colony formation assay showing Giemsa stained colonies ofMDAMB231 cells either in control or in chimera 4C5 treated cultures.FIG. 7B is a graphical presentation of the effect of the chimera 4C5 onthe number of cells in each colony. A 47.3% reduction is observed(p<0.001) when compared to control cultures.

FIG. 8 shows that monoclonal Ab 4C5 inhibits the metastatic depositionof breast cancer_([PC3]) cells into the lungs of SCID mice.

FIG. 9 shows that monoclonal Ab 4C5 inhibits the metastasis oftriple-negative breast cancer cells into the lungs of SCID mice.

FIG. 10 shows that monoclonal Ab 4C5 delays primary tumor growth ofMDAMB231 triple negative breast cancer cells growing orthotopically inSCID mice.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

By “mAb 4C5” is meant a polypeptide having at least 75%, 85%, 90%, 95%or even 99% amino acid sequence identity to SEQ ID NO: 2, or a fragmentthereof having antineoplastic activity and/or having HSP90-specificbinding activity. mAb 4C5 is a murine antibody that specificallyrecognizes both the α and to a lesser extent the β isoforms of humanheat shock protein 90 (HSP90).

The sequence of SEQ ID NO: 2 is provided below:

ELVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTINSLEYEDMGIYYCLQYDEFPRLTFGAGTRLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK

By “chimeric 4C5 protein” is meant a polypeptide having at least 75%,85%, 90%, 95% or even 99% amino acid sequence identity to the amino acidsequence of SEQ ID NO: 4, or a fragment thereof having antineoplasticactivity and/or having HSP90-specific binding activity. The human-mousechimeric 4C5 antibody was constructed by replacing the mouse mAb 4C5C_(K) with the corresponding human C_(K) domain. The human-mousechimeric 4C5ΔCys protein was constructed by deleting the very lastamino-acid (cysteine) of the human-mouse chimeric 4C5 antibody.

The sequence of SEQ ID NO: 4 is provided below:

ELVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTINSLEYEDMGIYYCLQYDEFPRLTFGAGTRLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

By “chimeric 4C5 polynucleotide” is meant any nucleic acid moleculeencoding a chimeric 4C5 polypeptide.

By “4C5 CDR1” is meant a polypeptide having at least 75%, 85%, 90%, 95%or even 99% amino acid sequence identity to SEQ ID NO: 5, wherein anantibody comprising 4C5 CDR1 has HSP90-specific binding activity. Thesequence of SEQ ID NO: 5 is provided below:

KASQDINSYLS.

By “4C5 CDR2” is meant a polypeptide having at least 75%, 85%, 90%, 95%or even 99% amino acid sequence identity to SEQ ID NO: 6, wherein anantibody comprising 4C5 CDR2 has HSP90-specific binding activity. Thesequence of SEQ ID NO: 6 is provided below:

RANRLVD.

By “4C5 CDR3” is meant a polypeptide having at least 75%, 85%, 90%, 95%or even 99% amino acid sequence identity to SEQ ID NO: 7, wherein anantibody comprising 4C5 CDR3 has HSP90-specific binding activity. Thesequence of SEQ ID NO: 7 is provided below:

LQYDEFPRLT.

By “HSP90-specific binding activity” is meant that an antibody of theinvention specifically binds to an HSP90 polypeptide.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease. By“analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “chimeric antibody” is meant an antibody comprising at least twodiscrete polypeptide fragments, a first polypeptide fragment from amurine antibody and a second polypeptide fragment from a human antibody.Each of the first and second polypeptide fragments are encoded by anucleic acid construct and are operatively linked such that uponexpression of the construct, a functional chimeric antibody comprisingthe murine antibody fragment linked to a human antibody fragment isgenerated. In one embodiment, the murine antibody fragment comprises oneor more complementarity determining regions each of which specificallybinds HSP90.

The term “cytotoxic moiety” includes, but is not limited to, abrin,ricin, Pseudomonas exotoxin, diphtheria toxin, botulinum toxin, ormodified toxins thereof.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ. In oneembodiment, the disease is a cancer or neoplasia.

By “effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative to an untreated patient. The effectiveamount of active compound(s) used to practice the present invention fortherapeutic treatment of a disease varies depending upon the manner ofadministration, the age, body weight, and general health of the subject.Ultimately, the attending physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as an“effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, in certain embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of thereference nucleic acid molecule or polypeptide. A fragment may contain10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 nucleotides or amino acids.

By “functional moiety” is meant any compound, agent, molecule, etc.,that possesses an activity or property that alters, enhances, orotherwise changes the ability of the targeting agent to fulfill anyparticular purpose or that enables the targeting agent to fulfill a newpurpose. Such purposes include, but are not limited to, providingdiagnostic and/or prognostic information and/or treatment of diseases orconditions associated with neoplasia.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. In certain embodiments, thepreparation is at least 75%, at least 90%, and even at least 99%, byweight, a polypeptide of the invention. An isolated polypeptide of theinvention may be obtained, for example, by extraction from a naturalsource, by expression of a recombinant nucleic acid encoding such apolypeptide; or by chemically synthesizing the protein. Purity can bemeasured by any appropriate method, for example, column chromatography,polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neoplasia” is meant a disease characterized by the pathologicalproliferation of a cell or tissue and its subsequent migration to orinvasion of other tissues or organs. Neoplasia growth is typicallyuncontrolled and progressive, and occurs under conditions that would notelicit, or would cause cessation of, multiplication of normal cells.

Neoplasias can affect a variety of cell types, tissues, or organs,including but not limited to an organ selected from the group consistingof bladder, bone, brain, breast, cartilage, glia, esophagus, fallopiantube, gallbladder, heart, intestines, kidney, liver, lung, lymph node,nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin,spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, and vagina, or a tissue orcell type thereof. Neoplasias include cancers, such as sarcomas,carcinomas, or plasmacytomas (malignant tumor of the plasma cells). Inone embodiment, a neoplasia is breast cancer or melanoma.

Neoplasia cells that invade surrounding tissue or enter the bloodstreamor lymphatic vessels form secondary tumors, or metastases, at a distancefrom the original tumor. Neoplasia that has metastasized is moredifficult to treat and often has a poorer prognosis. Depending on theseverity of the neoplasia (i.e., tumor size and invasiveness), a stagenumber is assigned, I, II, III, or IV. Stage I neoplasias are the leastadvanced and have the best prognosis. Stage II neoplasias typicallyinclude larger tumors and are associated with a somewhat poorerprognosis. Stage III and IV neoplasias have spread beyond their sites oforigin and have the poorest prognosis.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

As used herein, “recombinant” includes reference to a polypeptideproduced using cells that express a heterologous polynucleotide encodingthe polypeptide. The cells produce the recombinant polypeptide becausethey have been genetically altered by the introduction of theappropriate isolated nucleic acid sequence. The term also includesreference to a cell, or nucleic acid, or vector, that has been modifiedby the introduction of a heterologous nucleic acid or the alteration ofa native nucleic acid to a form not native to that cell, or that thecell is derived from a cell so modified. Thus, for example, recombinantcells express genes that are not found within the native(non-recombinant) form of the cell, express mutants of genes that arefound within the native form, or express native genes that are otherwiseabnormally expressed, under-expressed or not expressed at all.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, at least about20 amino acids, at least about 25 amino acids, and in certainembodiments, about 35 amino acids, about 50 amino acids, or about 100amino acids. For nucleic acids, the length of the reference nucleic acidsequence will generally be at least about 50 nucleotides, at least about60 nucleotides, at least about 75 nucleotides, and even about 100nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1%) SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). In certain embodiments, such asequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical atthe amino acid level or nucleic acid to the sequence used for comparison(i.e., to a reference sequence). By “specifically binds” is meant acompound or antibody that recognizes and binds a polypeptide of theinvention, but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample, which naturallyincludes a polypeptide of the invention.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

A “tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof. Any compositions or methods provided herein can be combinedwith one or more of any of the other compositions and methods providedherein.

The invention provides polypeptides which possess anti-tumor properties.The polypeptides described herein include mouse-human chimerascorresponding to antibody light chains, that lack antibody heavy chains,yet retain the ability to bind to HSP90α protein.

The invention is based, at least in part, on the discovery that themonoclonal antibody 4C5 is completely devoid of a heavy chain andconsists of a functional kappa light chain dimer, and the producing ofrecombinant chimeric mouse-human antibody light chain that retains theoriginal antibody's specificity and functional properties. Inparticular, the chimeric antibody inhibited the function of surfaceHSP90 and reduced breast and melanoma cancer cell invasion in vitro,reduced the metastatic deposits and metastasis of triple-negative breastcancer cells into the lungs of SCID mice and delayed the primary tumorgrowth of MDAMB231 triple negative breast cancer cells growingorthotopically in SCID mice. These results indicate that this chimericantibody is useful as an anti-cancer agent, with reduced adverseimmunogenic effects.

Accordingly, the invention provides therapeutic compositions comprisingmouse-human chimeric antibodies, and methods of using such antibodies toprevent, reduce, or eliminate the invasiveness of neoplastic cells(e.g., breast cancer and melanoma cells) or to otherwise treat aneoplasia or symptom thereof.

Antibodies

Antibodies (e.g., chimeric 4C5) that selectively bind an anti-HSP90polypeptide are useful in the methods of the invention. Such antibodiesare particularly useful for reducing or eliminating the growth andinvasiveness of a neoplastic cell. In particular, such antibodies may beused to reduce or eliminate the development and metastatic potential ofa tumor. As described herein below, binding to the HSP90 polypeptidereduces HSP90 biological activity in a neoplastic cell as assayed byanalyzing the colony-forming ability and invasiveness of neoplasticcells. In some embodiments the colony-forming ability is tested byanalyzing the growth of neoplastic cell colonies in vitro using aclonogenic assay. In certain embodiments, invasiveness is assayed invitro by detecting migration of a neoplastic cell into a wound inculture, by detecting actin rearrangement, or by detecting thedevelopment of lamellipodia. In other embodiments, invasiveness isassayed by detecting the metastasis of a neoplastic cell in an animalmodel in vivo. Methods of preparing antibodies are well known to thoseof ordinary skill in the science of immunology. As used herein, the term“antibody” means not only intact antibody molecules, but also fragmentsof antibody molecules that retain immunogen-binding ability. Suchfragments are also well known in the art and are regularly employed bothin vitro and in vivo. Accordingly, as used herein, the term “antibody”means not only intact immunoglobulin molecules, but also the well-knownactive fragments F(ab′)2, and Fab. F(ab′)2, and Fab fragments that lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983). In certainembodiments, the antibodies of the invention comprise chimericantibodies, humanized antibodies, fusion polypeptides, andunconventional antibodies. In other embodiments, the invention provideshybrid antibodies, in which one portion of the antibody is obtained froma first antibody (e.g., a murine antibody), while the other portion isobtained from a different second antibody (e.g., human antibody). Suchantibodies are often referred to as “chimeric” antibodies. Such hybridsmay also be formed using humanized antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc regionhas been enzymatically cleaved, or which has been produced without theFc region, designated an “F(ab)₂” fragment, retains both of the antigenbinding sites of the intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an “Fab” fragment, retains one of theantigen binding sites of the intact antibody. Fab fragments consist of acovalently bound antibody light chain and a portion of the antibodyheavy chain, denoted “Fd.” The Fd fragments are the major determinantsof antibody specificity (a single Fd fragment may be associated with upto ten different light chains without altering antibody specificity).Isolated Fd fragments retain the ability to specifically bind toimmunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizingHSP90, or immunogenic fragments thereof, as an immunogen. One method ofobtaining antibodies is to immunize suitable host animals with animmunogen and to follow standard procedures for polyclonal or monoclonalantibody production. The immunogen will facilitate presentation of theimmunogen on the cell surface. Immunization of a suitable host can becarried out in a number of ways. Nucleic acid sequences encoding a HSP90polypeptide or immunogenic fragments thereof, can be provided to thehost in a delivery vehicle that is taken up by immune cells of the host.The cells will in turn express the receptor on the cell surfacegenerating an immunogenic response in the host. Alternatively, nucleicacid sequences encoding a HSP90 polypeptide, or immunogenic fragmentsthereof, can be expressed in cells in vitro, followed by isolation ofthe polypeptide and administration of the polypeptide to a suitable hostin which antibodies are raised.

Alternatively, antibodies against a HSP90 polypeptide may, if desired,be derived from an antibody phage display library. A bacteriophage iscapable of infecting and reproducing within bacteria, which can beengineered, when combined with human antibody genes, to display humanantibody proteins. Phage display is the process by which the phage ismade to ‘display’ the human antibody proteins on its surface. Genes fromthe human antibody gene libraries are inserted into a population ofphage. Each phage carries the genes for a different antibody and thusdisplays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified fromthe host. Antibody purification methods may include salt precipitation(for example, with ammonium sulfate), ion exchange chromatography (forexample, on a cationic or anionic exchange column run at neutral pH andeluted with step gradients of increasing ionic strength), gel filtrationchromatography (including gel filtration HPLC), and chromatography onaffinity resins such as protein A, protein G, hydroxyapatite, andantiimmunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineeredto express the antibody. Methods of making hybridomas are well known inthe art. The hybridoma cells can be cultured in a suitable medium, andspent medium can be used as an antibody source. Polynucleotides encodingthe antibody of interest can in turn be obtained from the hybridoma thatproduces the antibody, and then the antibody may be producedsynthetically or recombinantly from these DNA sequences. For theproduction of large amounts of antibody, it is generally more convenientto obtain an ascites fluid. The method of raising ascites generallycomprises injecting hybridoma cells into an immunologically naivehistocompatible or immunotolerant mammal, especially a mouse. The mammalmay be primed for ascites production by prior administration of asuitable composition (e.g., Pristane).

Antibodies produced by methods of the invention can be “humanized” bymethods known in the art. “Humanized” antibodies are antibodies in whichat least part of the sequence has been altered from its initial form torender it more like human immunoglobulins. Techniques to humanizeantibodies are particularly useful when non-human animal (e.g., murine)antibodies are generated. Examples of methods for humanizing a murineantibody are provided in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,225,539,5,585,089, 5,693,762 and 5,859,205. In one embodiment, the heavy chainand light chain C regions are replaced with human sequence. In anotherversion, the CDR regions comprise amino acid sequences for recognitionof an antigen of interest, while the variable framework regions havealso been converted to human sequences. It is well established thatnon-CDR regions of a mammalian antibody may be replaced withcorresponding regions of non-specific or hetero-specific antibodieswhile retaining the epitope specificity of the original antibody. Thistechnique is useful for the development and use of humanized antibodiesin which non-human CDRs are covalently joined to human FR and/or Fc/pFc′regions to produce a functional antibody. In a third version, variableregions are humanized by designing consensus sequences of human andmouse variable regions, and converting residues outside the CDRs thatare different between the consensus sequences.

In one embodiment humanized antibodies are generated by operably linkingnucleic acid sequences encoding the amino acids comprising the CDRs (SEQID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7) to nucleic acid sequencesencoding human Frame work region sequences (FR1, FR2, and FR3).

In other embodiments, the invention provides “unconventionalantibodies.”

Unconventional antibodies include, but are not limited to, nanobodies,linear antibodies (Zapata et al, Protein Eng. 8(10): 1057-1062, 1995),single domain antibodies, single chain antibodies, and antibodies havingmultiple valencies (e.g., diabodies, tribodies, tetrabodies, andpentabodies). Nanobodies are the smallest fragments of naturallyoccurring heavy-chain antibodies that have evolved to be fullyfunctional in the absence of a light chain. Nanobodies have the affinityand specificity of conventional antibodies although they are only halfof the size of a single chain Fv fragment. The consequence of thisunique structure, combined with their extreme stability and a highdegree of homology with human antibody frameworks, is that nanobodiescan bind therapeutic targets not accessible to conventional antibodies.Recombinant antibody fragments with multiple valencies provide highbinding avidity and unique targeting specificity to cancer cells. Thesemultimeric scFvs (e.g., diabodies, tetrabodies) offer an improvementover the parent antibody since small molecules of ˜60-100 kDa in sizeprovide faster blood clearance and rapid tissue uptake See Power et al.,(Generation of recombinant multimeric antibody fragments for tumordiagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu etal. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumortargeting and imaging. Tumor Targeting, 4, 47-58, 1999).

Various techniques for making unconventional antibodies have beendescribed.

Bispecific antibodies produced using leucine zippers are described byKostelny et al. (J. Immunol. 148(5): 1547-1553, 1992). Diabodytechnology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA90:6444-6448, 1993). Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers is described byGruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies aredescribed by Tutt et al. (J. Immunol. 147:60, 1991). Single chain Fvpolypeptide antibodies include a covalently linked V_(H)::V_(L)heterodimer which can be expressed from a nucleic acid including V_(H)-and V_(L)-encoding sequences either joined directly or joined by apeptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad.Sci. USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513,5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754and 20050196754.

Antibodies of the invention are particularly useful for the treatment ofneoplasias, including breast cancer and melanoma, or other tumorsincluding but not limited to gioblastomas, lymphomas, colon cancer,non-small cell lung cancer etc. Accordingly, the present inventionprovides methods of treating neoplastic disease and/or disorders orsymptoms thereof which comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising a chimericantibody described herein to a subject (e.g., a mammal such as a human).Thus, one embodiment is a method of treating a subject suffering from orsusceptible to a neoplastic disease or disorder or symptom thereof. Themethod includes the step of administering to the mammal a therapeuticamount (of an amount) of a compound herein sufficient to treat thedisease or disorder or symptom thereof, under conditions such that thedisease or disorder is treated. The methods herein include administeringto the subject (including a subject identified as in need of suchtreatment) a therapeutically effective amount of a compound describedherein, or a composition described herein to produce such effect.Identifying a subject in need of such treatment can be in the judgmentof a subject or a health care professional and can be subjective (e.g.opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the chimeric antibodies delineated herein to asubject (e.g. animal, human) in need thereof, including a mammal,particularly a human. Such treatment will be suitably administered tosubjects, particularly humans, suffering from, having, susceptible to,or at risk for metastasis in connection with a neoplastic disease.Determination of those subjects “at risk” can be made by any objectiveor subjective determination by a diagnostic test or opinion of a subjector health care provider (e.g., genetic test, enzyme or protein marker,Marker (as defined herein), family history, and the like).

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withneoplasia, in which the subject has been administered a therapeuticamount of a compound herein sufficient to treat the disease or symptomsthereof. The level of Marker determined in the method can be compared toknown levels of Marker in either healthy normal controls or in otherafflicted patients to establish the subject's disease status. In certainembodiments, a second level of Marker in the subject is determined at atime point later than the determination of the first level, and the twolevels are compared to monitor the course of disease or the efficacy ofthe therapy. In certain embodiments, a pre-treatment level of Marker inthe subject is determined prior to beginning treatment according to thisinvention; this pre-treatment level of Marker can then be compared tothe level of Marker in the subject after the treatment commences, todetermine the efficacy of the treatment.

Recombinant Polypeptide Expression

The invention provides antibodies (chimeric antibodies) of the inventionthat are useful for the treatment of neoplasias. In particular, theinvention provides a human-mouse chimeric 4C5 antibody, which can beexpressed recombinantly. Typically, recombinant polypeptides areproduced by transformation of a suitable host cell with all or part of apolypeptide-encoding nucleic acid molecule or fragment thereof in asuitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A polypeptide of the invention may be produced in aprokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g.,Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammaliancells, e.g., NIH 3T3, HeLa, or COS cells). Such cells are available froma wide range of sources (e.g., the American Type Culture Collection,Rockland, Md.; also, see, e.g., Ausubel et al, Current Protocol inMolecular Biology, New York: John Wiley and Sons, 1997). The method oftransformation or transfection and the choice of expression vehicle willdepend on the host system selected. Transformation and transfectionmethods are described, e.g., in Ausubel et al. (supra); expressionvehicles may be chosen from those provided, e.g., in Cloning Vectors: ALaboratory Manual (P. H. Pouwels et al, 1985, Supp. 1987).

A variety of expression systems exist for the production of thepolypeptides of the invention. Expression vectors useful for producingsuch polypeptides include, without limitation, chromosomal, episomal,and virus-derived vectors, e.g., vectors derived from bacterialplasmids, from bacteriophage, from transposons, from yeast episomes,from insertion elements, from yeast chromosomal elements, from virusessuch as baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof.

One particular bacterial expression system for polypeptide production isthe E. coli pET expression system (e.g., pET-28) (Novagen, Inc.,Madison, Wis.). According to this expression system, DNA encoding apolypeptide is inserted into a pET vector in an orientation designed toallow expression. Since the gene encoding such a polypeptide is underthe control of the T7 regulatory signals, expression of the polypeptideis achieved by inducing the expression of T7 RNA polymerase in the hostcell. This is typically achieved using host strains that express T7 RNApolymerase in response to IPTG induction. Once produced, recombinantpolypeptide is then isolated according to standard methods known in theart, for example, those described herein.

Another bacterial expression system for polypeptide production is thepGEX expression system (Pharmacia). This system employs a GST genefusion system that is designed for high-level expression of genes orgene fragments as fusion proteins with rapid purification and recoveryof functional gene products. The protein of interest is fused to thecarboxyl terminus of the glutathione S-transferase protein fromSchistosoma japonicum and is readily purified from bacterial lysates byaffinity chromatography using Glutathione Sepharose 4B. Fusion proteinscan be recovered under mild conditions by elution with glutathione.Cleavage of the glutathione S-transferase domain from the fusion proteinis facilitated by the presence of recognition sites for site-specificproteases upstream of this domain. For example, proteins expressed inpGEX-2T plasmids may be cleaved with thrombin; those expressed inpGEX-3× may be cleaved with factor Xa.

Alternatively, recombinant polypeptides of the invention are expressedin Pichia pastoris, a methylotrophic yeast. Pichia is capable ofmetabolizing methanol as the sole carbon source. The first step in themetabolism of methanol is the oxidation of methanol to formaldehyde bythe enzyme, alcohol oxidase. Expression of this enzyme, which is codedfor by the AOX1 gene is induced by methanol. The AOX1 promoter can beused for inducible polypeptide expression or the GAP promoter forconstitutive expression of a gene of interest.

Once the recombinant polypeptide of the invention is expressed, it isisolated, for example, using affinity chromatography. In one example, anantibody (e.g., produced as described herein) raised against apolypeptide of the invention may be attached to a column and used toisolate the recombinant polypeptide. Lysis and fractionation ofpolypeptide-harboring cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al, supra).Alternatively, the polypeptide is isolated using a sequence tag, such asa hexahistidine tag, that binds to nickel column.

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry and Molecular Biology,eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention,particularly short peptide fragments, can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, 111). Thesegeneral techniques of polypeptide expression and purification can alsobe used to produce and isolate useful peptide fragments or analogs(described herein).

Antibodies and Analogs Thereof

The invention further provides antibodies (e.g., human/mouse chimericantibodies) or fragments thereof that are modified in ways that enhanceor do not inhibit their ability to reduce the growth, proliferation, orsurvival of a neoplastic cell. In one embodiment, the invention providesmethods for optimizing the amino acid sequence of the chimeric antibodyor the nucleic acid sequence encoding the chimeric antibody by producingan alteration. Such changes may include certain mutations, deletions,insertions, or post-translational modifications. In one embodiment, theamino acid sequence is modified to enhance protease resistance.Accordingly, the invention further includes analogs of anynaturally-occurring polypeptide of the invention. Analogs can differfrom the naturally-occurring the polypeptide of the invention by aminoacid sequence differences, by post-translational modifications, or byboth. Analogs of the invention will generally exhibit at least 75%,85%>, 90%, 95% or even 99% identity with all or part of anaturally-occurring amino, acid sequence of the invention. The length ofsequence comparison is at least 10, 13, 15 amino acid residues, at least25 amino acid residues, and more than 35 amino acid residues. Again, inan exemplary approach to determining the degree of identity, a BLASTprogram may be used, with a probability score between e⁻³ and e⁻¹⁰⁰indicating a closely related sequence. Modifications include in vivo andin vitro chemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation; such modifications mayoccur during polypeptide synthesis or processing or following treatmentwith isolated modifying enzymes. Analogs can also differ from thenaturally-occurring polypeptides of the invention by alterations inprimary sequence. These include genetic variants, both natural andinduced (for example, resulting from random mutagenesis by irradiationor exposure to ethanemethylsulfate or by site-specific mutagenesis asdescribed in Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al, supra).Also included are cyclized peptides, molecules, and analogs whichcontain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids.

In addition to full-length polypeptides, the invention also includesfragments of any one of the polypeptides of the invention. As usedherein, the term “a fragment” means at least 5, 10, 13, or 15 aminoacids in length. In other embodiments a fragment is at least 20contiguous amino acids, at least 30 contiguous amino acids, or at least50 contiguous amino acids, and in other embodiments at least 60 to 80 ormore contiguous amino acids. Fragments of the invention can be generatedby methods known to those skilled in the art or may result from normalprotein processing (e.g., removal of amino acids from the nascentpolypeptide that are not required for biological activity or removal ofamino acids by alternative mRNA splicing or alternative proteinprocessing events).

Antibody analogs having a chemical structure designed to mimic antibodyfunctional activity (e.g., anti-neoplastic activity, antigen bindingactivity) can be administered according to methods of the invention.Methods of analog design are well known in the art, and synthesis ofanalogs can be carried out according to such methods by modifying thechemical structures such that the resultant analogs exhibit theantineoplastic activity of SEQ ID NO:2 or a chimeric antibody comprisingSEQ ID NO:4_([PC6]). In certain embodiments, the antibody analogs arerelatively resistant to in vivo degradation, resulting in a moreprolonged therapeutic effect upon administration.

Assays for measuring functional activity include, but are not limitedto, those described in the Examples below.

Pharmaceutical Therapeutics

The invention chimeric antibodies that are useful for the treatment ofneoplasia. In one particular embodiment, the chimeric antibodies of theinvention are useful for preventing or reducing tumor growth and thepropensity of a neoplastic cell to invade a surrounding tissue or tootherwise metastasize. For therapeutic uses, the antibodies disclosedherein may be administered systemically, for example, formulated in apharmaceutically-acceptable buffer such as physiological saline. Incertain embodiments, routes of administration include, for example,subcutaneous, intravenous, interperitoneally, intramuscular, orintradermal injections that provide continuous, sustained levels of thedrug in the patient. Treatment of human patients or other animals willbe carried out using a therapeutically effective amount of a therapeuticidentified herein in a physiologically-acceptable carrier. Suitablecarriers and their formulation are described, for example, inRemington's Pharmaceutical Sciences by E. W. Martin. The amount of thetherapeutic agent to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with theclinical symptoms of the neoplasia. Generally, amounts will be in therange of those used for other agents used in the treatment of otherdiseases associated with neoplasia, although in certain instances loweramounts will be needed because of the increased specificity of thecompound.

Therapeutic Methods

Antibodies of the invention (e.g., murine and human-murine chimericantibodies described herein) are useful for preventing or amelioratingneoplastic disease. In one therapeutic approach, an agent identified ordescribed herein is administered to the site of a potential or actualdisease-affected tissue or is administered systemically. The dosage ofthe administered agent depends on a number of factors, including thesize and health of the individual patient. For any particular subject,the specific dosage regimes should be adjusted over time according tothe individual need and the professional judgment of the personadministering or supervising the administration of the compositions.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of neoplasia may beby any suitable means that results in a concentration of the therapeuticthat, combined with other components, is effective in ameliorating,reducing, or stabilizing a neoplasia. The compound may be contained inany appropriate amount in any suitable carrier substance, and isgenerally present in an amount of 1-95% by weight of the total weight ofthe composition. The composition may be provided in a dosage form thatis suitable for parenteral (e.g., subcutaneously, intravenously,intramuscularly, or intraperitoneally) administration route. Anadvantageous method of administration is intravenous infusion. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the antibody substantially immediately upon administration orat any predetermined time or time period after administration. Thelatter types of compositions are generally known as controlled releaseformulations, which include (i) formulations that create a substantiallyconstant concentration of the drug within the body over an extendedperiod of time; (ii) formulations that after a predetermined lag timecreate a substantially constant concentration of the drug within thebody over an extended period of time; (iii) formulations that sustainaction during a predetermined time period by maintaining a relatively,constant, effective level in the body with concomitant minimization ofundesirable side effects associated with fluctuations in the plasmalevel of the active substance (sawtooth kinetic pattern); (iv)formulations that localize action by, e.g., spatial placement of acontrolled release composition adjacent to or in contact with thethymus; (v) formulations that allow for convenient dosing, such thatdoses are administered, for example, once every one or two weeks; and(vi) formulations that neoplasia by using carriers or chemicalderivatives to deliver the therapeutic agent to a particular cell type(e.g., neoplastic cell). For some applications, controlled releaseformulations obviate the need for frequent dosing during the day tosustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that reduces orameliorates a neoplasia, the composition may include suitableparenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable therapeutic(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution and dextrose solution.The aqueous formulation may also contain one or more preservatives(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where oneof the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the antibodymay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices. Materials for use in the preparation ofmicrospheres and/or microcapsules are, e.g., biodegradable/bioerodiblepolymers such as polygalactia poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid).

Biocompatible carriers that may be used when formulating a controlledrelease parenteral formulation are carbohydrates (e.g., dextrans),proteins (e.g., albumin), lipoproteins, or antibodies. Materials for usein implants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. Such formulations are known to the skilled artisan.Excipients may be, for example, inert diluents or fillers (e.g.,sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starchesincluding potato starch, calcium carbonate, sodium chloride, lactose,calcium phosphate, calcium sulfate, or sodium phosphate); granulatingand disintegrating agents (e.g., cellulose derivatives includingmicrocrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material, such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the chimeric antibody). The coatingmay be applied on the solid dosage form in a similar manner as thatdescribed in Encyclopedia of Pharmaceutical Technology, supra.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructedto release the chimeric antibody therapeutic by controlling thedissolution and/or the diffusion of the active substance. Dissolution ordiffusion controlled release can be achieved by appropriate coating of atablet, capsule, pellet, or granulate formulation of compounds, or byincorporating the compound into an appropriate matrix. A controlledrelease coating may include one or more of the coating substancesmentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax,carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryldistearate, glycerol palmitostearate, ethylcellulose, acrylic resins,dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride,polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3butylene glycol, ethylene glycol methacrylate, and/or polyethyleneglycols. In a controlled release matrix formulation, the matrix materialmay also include, e.g., hydrated methylcellulose, carnauba wax andstearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more therapeuticcompounds may also be in the form of a buoyant tablet or capsule (i.e.,a tablet or capsule that, upon oral administration, floats on top of thegastric content for a certain period of time). A buoyant tabletformulation of the compound(s) can be prepared by granulating a mixtureof the compound(s) with excipients and 20-75% w/w of hydrocolloids, suchas hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Combination Therapies

Optionally, chimeric antibody therapeutics of the invention may beadministered in combination with any other chemotherapeutic; suchmethods are known to the skilled artisan and described in Remington'sPharmaceutical Sciences by E. W. Martin.

Kits

The invention provides kits for the treatment or prevention ofneoplasia. In one embodiment, the kit includes a therapeutic orprophylactic composition containing a therapeutically effective amountof a chimeric antibody in unit dosage form. In some embodiments, the kitcomprises a sterile container which contains a therapeutic orprophylactic cellular composition; such containers can be boxes,ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or othersuitable container forms known in the art. Such containers can be madeof plastic, glass, laminated paper, metal foil, or other materialssuitable for holding medicaments.

If desired a chimeric antibody of the invention is provided togetherwith instructions for administering the chimeric antibody to a subjecthaving or at risk of developing cancer (e.g., melanoma, breast cancer).The instructions will generally include information about the use of thecomposition for the treatment or prevention of neoplasia. In otherembodiments, the instructions include at least one of the following:description of the therapeutic agent; dosage schedule and administrationfor treatment or prevention of ischemia or symptoms thereof;precautions; warnings; indications; counter-indications; overdosageinformation; adverse reactions; animal pharmacology; clinical studies;and/or references. The instructions may be printed directly on thecontainer (when present), or as a label applied to the container, or asa separate sheet, pamphlet, card, or folder supplied in or with thecontainer.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 MAb 4C5 Lacks a Heavy Chain

The electrophoretic motility of mAb 4C5, which was isolated fromhybridoma cultures, was studied under reducing and non-reducingSDS-PAGE. This analysis revealed that this murine monoclonal antibody isnot a conventional IgG molecule. When purified mAb 4C5 was subjected toreducing SDS-PAGE, followed by immunoblotting with an anti-mouse Fabantibody, the typical 25 kDa and 50 kDa bands, corresponding to theantibody light (L-) and heavy (H-) chains respectively, of aconventional IgG antibody were not observed. Instead a single band ofapproximately 25 kDa was seen (FIG. 1A). Interestingly, an identical 25kDa single band was obtained after immunoblotting with an anti-kappaL-chain antibody (FIG. 1A). Accordingly, after non-reducingelectrophoresis followed by immunoblotting with both the above mentionedantibodies, mAb 4C5 was shown to be significantly smaller than aconventional IgG1 molecule since it migrated at approximately 50 kDainstead of 150 kDa (FIG. 1A). Finally, no immunoreactivity was detectedafter electrophoresis of mAb 4C5 under both reducing and non-reducingconditions, followed by western blot analysis using an anti-Fcγantibody(FIG. 1A). Taken together, these data indicated that mAb 4C5 eitherlacks a part of its H-chain, or that it is completely devoid of aH-chain.

To further explore these possibilities, northern blot analysis as wellas PCR amplification of the IgG1 H-chain cDNA was performed. In contrastto the positive control, no radioactivity was detected when RNAs derivedfrom the mAb 4C5 hybridoma and NSO myeloma cells (negative control) werehybridized with the H-chain probe (FIG. 1B). These results indicatedthat mAb 4C5 likely completely lacks an H-chain gene. This was furtherconfirmed by the H-chain PCR amplification experiments. For theamplification of the H-chain cDNA of mAb 4C5, a panel of 8 mouseuniversal primers and a polyA+ primer directed against the 5′ and the 3′of the gene, respectively, were tested in several separate PCRreactions. In all conditions tested no amplification of a specificH-chain PCR product was observed. These combined data demonstrate thatmAb 4C5 is devoid of an H-chain.

Example 2 A Human-mouse Chimeric Antibody was Constructed

The cDNA encoding mAb 4C5 was isolated from 4C5 expressing hybridomas.The initial amplification of the mAb 4C5 kappa chain gene from the firststrand cDNA template was performed using universal mouse L-chainprimers. The approximately 650 by PCR product corresponding to the fulllength mAb 4C5 L-chain, was then subcloned into the SacI and Xbalrestriction sites of the pComb3HSS_([PC7]) vector. The sequence analysisof cloned mAb 4C5 L-chain revealed that it belongs to the kappa chainsubgroup I (FIG. 2).

A human-mouse chimeric 4C5 antibody was constructed by replacing themouse mAb 4C5 C_(K) with the corresponding human C_(K) domain. Thesequence analysis of several recombinant clones confirmed the successfulconstruction of the chimeric mouse-human 4C5. Both recombinant 4C5 andchimeric 4C5 antibodies were expressed in soluble forms in bacterialsupernatants and purified. The electrophoretic motilities of thepurified antibodies were tested with SDS-PAGE under reducing andnon-reducing conditions and were found to be similar with thecorresponding motility of the original mAb 4C5 antibody (FIG. 3).

To improve the translational efficacy of ch4C5 and in bacterialexpression systems, standard molecular biology techniques known in theart were used to mutate the cDNA sequence of ch4C5 (SEQ ID NO: 3) at thewobble position, such as to remove rare tRNA codons (e.g., see Kane1995: Effects of rare codon clusters on high-level expression ofheterologous proteins in Escherichia coli, Curr Opin Biotechnol. 1995October; 6(5):494-500). SEQ ID NO: 9 exhibits a cDNA sequence of ch4C5that is optimized for rare codon usage, leading to an improved yield inbacterial expression. Thus, ch4C5 can be expressed by usage of SEQ IDNO: 3, preferably by SEQ ID NO: 9. In addition, a chimeric 4C5 deletionmutant, referred to herein as ch4C5ΔCys, was constructed by removing thelast three nucleotides of SEQ ID NO: 9, namely the triplet “TGC”encoding for cystein, to create SEQ ID: NO 10. Thus, ch4C5ΔCys can beexpressed by usage of SEQ ID NO: 10 and the ch4C5ΔCys is identical toSEQ ID NO: 4 but for the last cysteine at the 3′ end.

Purified chimeric 4C5 and ch4C5ΔCys antibodies expressed in bacteria asdisclosed herein were analyzed with SDS-PAGE as shown in FIG. 3C: lanes1-2 and 7-8: chimeric 4C5; lanes 3-4 and 9-10: ch4C5ΔCys; odd lanescorrespond to 2 μg of loaded antibody, whereas even lanes to 5 μg,except from lanes 5 (molecular weight control and negative control,respectively). The monoclonal antibody ch4C5ΔCys migrated as a monomerunder reducing or non-reducing conditions, suggesting that it existspredominantly as a monomer (FIG. 3C) Chimeric 4C5 migrated as a dimerunder non-reducing conditions and as a monomer under reducingconditions, suggesting that it may exist as a monomer or as a dimer(FIG. 3C).

Example 3 Chimeric 4C5 Specifically Recognized HSP90

In order to explore the specificity of the recombinant L-chains, westernblot analysis was performed in MDAMB453 breast cancer cell lysates usinga commercially available polyclonal anti-HSP90αantibody (obtained fromChemicon), mAb 4C5, recombinant 4C5, and chimeric 4C5. In all cases asingle identical immunoreactive band was observed (FIG. 4A), confirmingthat the mouse recombinant L-chain and the chimeric human-mouse L-chainretain the specificity of the paternal mAb 4C5. This was furtherconfirmed by immunoprecipitation experiments performed in pre-clearedMDAMB453 cell lysates using polyclonal anti-HSP90 a followed byimmunoblotting with mAb 4C5, recombinant 4C5, and chimeric 4C5. In allcases, a single immunoreactive band was observed, indicating that thechimeric light chain specifically recognized HSP90 (FIG. 4B). The sameresult was obtained when immunoprecipitation was performed using mAb4C5, recombinant 4C5, and chimeric 4C5 followed by western blotting withthe polyclonal anti-HSP90α antibody (FIG. 4C). In all experiments anirrelevant mouse IgG was used as a negative control.

Example 4 A Human/mouse Chimeric Light Chain Antibody Bound to the CellSurface and was not Internalized by MDAMB453 Cells

Previous studies have shown that mAb 4C5 specifically binds to surfaceHSP90. In order to investigate whether the chimeric 4C5 antibody alsoretains this feature, unfixed MDAMB453 cultures were incubated withrecombinant 4C5 and chimeric 4C5 L-chains, and after 2 hours in culture,the cells were carefully washed, fixed, and labelled withfluorescence-conjugated secondary antibody. Thus, the primary antibodyhad access only to the external surface of the cells. The observedtypical punctuate immunostaining confirmed the cell surface localizationof HSP90 (FIG. 5A). Similar results were obtained using commercialanti-HSP90α and of course mAb 4C5 (FIG. 5A). It is noteworthy thatsimilarly to mAb 4C5, chimeric 4C5 as well as recombinant 4C5 alsorecognized the intracellular HSP90 as demonstrated by immunofluoresenceafter fixation and permeabilization of MDAMB453 cells (FIG. 5B).Finally, the binding of both L-chain antibodies to living MDAMB453, wasmonitored at various time intervals. After incubation with theantibodies at 37° C., cells were fixed, permeabilized, and stained witha fluorescence-conjugated secondary antibody. It was shown that like tomAb 4C5 and in contrast to anti-HSP90α, recombinant and chimeric 4C5were not internalized, but remained bound on the cell surface (FIG. 5C).

Example 5 Chimeric Light Chain Did not Affect the Function ofIntracellular HSP90

The significance of the cell impermeability of the chimeric L-chainantibody was investigated by monitoring the levels of certain HSP90intracellular client proteins. To date more than one hundredintracellular HSP90 client proteins have been identified. Cell-permeableHSP90 inhibitors induce the degradation of these proteins because theirstability depends on intracellular HSP90 function. Because datademonstrated that mAb 4C5, recombinant 4C5, and chimeric 4C5 are alllikely to be cell impermeable, their ability to affect the stability ofthree well-characterized intracellular HSP90 client proteins (Akt, cRaf,and ErbB2) was examined.

MDAMB453 breast cancer cells were incubated with the indicatedconcentrations of either mAb 4C5, recombinant 4C5, or chimeric 4C5followed by monitoring of Akt, cRaf, and ErbB2 levels by westernblotting. As shown in FIG. 5D the antibodies tested did not affect thesteady-state levels of any of the HSP90 client proteins.

Example 6 Chimeric Light Chain Antibody Inhibited MDAMB453 and B16 F10Cancer Cell Invasion in a Wound Healing Assay

Previous studies have shown that mAb 4C5 inhibits MDAMB453 breast cancerand B 16 F10 melanoma cell invasion in a wound healing assay. In orderto determine whether the chimeric antibody exhibited the same functionalproperty as the parental mAb 4C5, in vitro wound healing assays wereperformed using MDAMB453 and B16 F10 cancer cells. As shown in FIGS. 6Aand 6B, the presence of chimeric 4C5 in the culture medium significantlyreduced the rate of MDAMB453 cancer cell invasion within the migrationgap after 48 hours, as compared to control cultures. The inhibition rateof MDAB453 invasion was similar to that obtained when the anti-HSP90α,as well as the parental mAb 4C5 and the recombinant 4C5 were included inthe culture medium (FIGS. 6A and B). Similar results were obtained in awound healing assay using B 16 F10 melanoma cells and increasingconcentrations of chimeric 4C5 (FIGS. 6C and 6D). Interestingly, theinhibition of melanoma cell invasion was dose dependent indicating thespecificity of the antibody (FIG. 6D). Control cultures were growneither in culture medium alone, or in culture medium containing 200μg/ml of an irrelevant IgG1 antibody. It is important to note that nostatistically significant difference was observed between the two typesof controls used. The control value illustrated is the mean value of thetwo types of control.

It should be noted that the inhibitory effect of the anti-HSP90αantibody on the invasion rate of the MDAMB453 cells was at least ingreat part due to the increased cell death as judged by trypan bluestaining (FIG. 6E). In contrast, when cultures were treated with mAb 4C5and chimeric 4C5, the cell death rate was similar to that observed inthe control cultures (FIG. 6E). This result further supports that thechimeric 4C5, in contrast to the polyclonal anti-HSP90αantibody, is notinternalized and thus does not affect the intracellular pool of HSP90which is important for cell survival.

The above described cultures were examined with respect to actinre-arrangement dynamics using fluorescently labelled phalloidin. Whencultures were exposed to the chimeric L-chain, less cell spreading wasobserved as compared to control cultures, and a morphology indicative ofnon-motile cells was seen. Furthermore, when these cultures werevisualized at a higher magnification, lamellipodia were less developedand spread out as compared to lamellipodia of MDAMB453 in controlcultures (FIGS. 6F and 6G). These results are in accordance topreviously published data regarding the effect of mAb 4C5 on actinre-arrangement and lamellipodia development.

Finally, FIG. 6H shows that ch4C5ΔCys inhibits B16F10 mouse melanomacell migration in an in vitro wound healing assay. Photographs representphase-contrast images obtained at zero time (left panel) and at 24 hours(right panel) after scratch formation, showing B16F10 mouse melanomacell migration either in control cultures or cultures includingch4C5ΔCys_([PC8]).

Example 7 Chimeric Light Chain Antibody Significantly Reduced the Numberof Cells in Each Individual Colony Formed by MDAMB231 Human BreastCancer Cells in a Clonogenic Assay

In order to investigate the capacity of the chimera 4C5 to affect thecapacity of MDAMB231 human breast cancer cells to form colonies aclonogenic assay was performed. When cultures were exposed to thechimeric L-chain a significant reduction of the number of cells in eachindividual colony was observed when compared to control cultures. Thisresult indicates that the chimeric antibody has a cytostatic effect onthe development of human breast cancer colonies.

The experiments reported herein describe the cloning and sequencing ofan anti-HSP90 murine monoclonal antibody, named mAb 4C5. Moreimportantly, it was found that mAb 4C5 is not a conventional IgGmolecule, but instead is completely devoid of a H-chain and consists ofa κ light chain dimer. This was initially supported by SDS-PAGEelectrophoresis under denaturing and non-denaturing conditions,demonstrating that mAb 4C5 migrated unconventionally at approximately26- and 50-kDa, respectively. Additionally, when total RNA isolated fromthe mAb 4C5-producing hybridoma cell line was subjected to northern blothybridization using an IgG1 H-chain cDNA radio-labeled probe, noradioactivity could be detected. In agreement with the above results,the H-chain cDNA could not be amplified using universal mouse H-chainprimers. Finally, the extra-ordinary nature of mAb 4C5 was confirmedbeyond any doubt, since the recombinant κ light chain expressed inbacteria was shown to retain all the properties of the paternalantibody, including antigen binding and in vitro inhibition of cancercell invasion.

This monoclonal antibody was originally produced by immunization of micewith a brain membrane fraction of 15-day-old rat embryos (Thomaidou D,Patsavoudi E. Identification of a novel neuron-specific surface antigenin the developing nervous system, by monoclonal antibody 4C5.Neuroscience. 1993; 53: 813-27), and it was shown to specificallyrecognize and inhibit the function of surface HSP90 during cellmigration processes (Sidera K, Samiotaki M, Yfanti E, Panayotou G,Patsavoudi E.

Involvement of cell surface HSP90 in cell migration reveals a novel rolein the developing nervous system. J Biol. Chem. 2004; 279: 45379-88).Additionally, mAb 4C5 was shown to significantly reduce the rate ofinvasion and metastasis of cancer cells. More specifically, it wasdemonstrated that this antibody inhibits melanoma cell invasion andmetastasis (Stellas D, Karameris A, Patsavoudi E. Monoclonal antibody4C5 immunostains human melanomas and inhibits melanoma cell invasion andmetastasis. Clin Cancer Res. 2007; 13: 1831-8) as well as theinteraction of surface HSP90 with the extracellular domain of HER-2,leading to impaired downstream signalling and subsequently reduced rateof in vitro breast cancer cell invasion (Sidera K, Gaitanou M, StellasD, Matsas R, Patsavoudi E. A critical role for HSP90 in cancer cellinvasion involves interaction with the extracellular domain of HER-2. JBiol. Chem. 2008; 283: 2031-41).

It is well known that murine antibodies have limited use for in vivotherapy in humans because of their immunogenicity. This problem has beenovercome using genetic engineering approaches to produce chimericmouse-human and fully human antibodies. Taking into consideration theunconventional nature of mAb 4C5 in combination with the fact thatduring the humanization process the antibody affinity is frequentlyreduced, the murine mAb was reconstituted into a functional mouse-humanchimeric version that, similarly to the paternal antibody binds tosurface HSP90 and inhibits cancer cell invasion. The chimeric antibodywhich was engineered by replacing the C-region of the paternal murineantibody with the corresponding human C-region, was shown to retain thespecificity and affinity of the paternal mouse antibody.

It has been a generally accepted conception that the antibody moleculerequires both the H- and L-chains for its full activity (Sastry L,Alting-Mees M, Huse W D, Short J M, Sorge J A, Hay B N, et al. Cloningof the immunological repertoire in Escherichia coli for generation ofmonoclonal catalytic antibodies: construction of a heavy chain variableregion-specific cDNA library. Proc Natl Acad Sci USA. 1989; 86:5728-32).

Moreover, the H-chain is believed to be the predominant contributor tothe free energy of binding while the contribution of the L-chain toantigen binding is supposed to be limited (Novotny J, Bruccoleri R E,Saul F A. On the attribution of binding energy in antigen-antibodycomplexes McPC 603, D1.3, and HyHEL-5. Biochemistry. 1989; 28: 4735-49;Ward E S, Gussow D, Griffiths A D, Jones P T, Winter G. Bindingactivities of a repertoire of single immunoglobulin variable domainssecreted from Escherichia coli. Nature. 1989; 341: 544-6). The latter isfurther supported by the fact that cameloids possesses a class of fullyfunctional antibodies completely lacking L-chains and consisting ofH-chain dimers (Hamers-Casterman C, Atarhouch T, Muyldermans S, RobinsonG, Hamers C, Songa E B, et al. Naturally occurring antibodies devoid oflight chains. Nature. 1993; 363: 446-8; Muyldermans S, Cambillau C, WynsL. Recognition of antigens by single-domain antibody fragments: thesuperfluous luxury of paired domains. Trends Biochem Sci. 2001; 26:230-5). In context to these findings, H-chains alone were shown tointeract with a variety of antigens in a specific manner (albeit withlower affinity than the intact antibodies), which has led to the use ofsingle domain antibodies derived from the H-chains (Winter G, MilsteinC. Man-made antibodies. Nature. 1991; 349: 293-9).

In the past, antigen binding by L-chains has been sporadicallydemonstrated. The first reports of free immunoglobulin light chainsconcerned the so called Bence-Jones proteins. These were reported asL-chain dimers expressed by multiple myeloma cells, collected from theurine of human patients (Bradwell A R, Carr-Smith H D, Mead G P, HarveyT C, Drayson M T. Serum test for assessment of patients with Bence Jonesmyeloma. Lancet. 2003; 361: 489-91). Furthermore, large amounts ofL-chains accumulate in the extracellular fluids and tissues of patientswith L-chain secreting tumors (Stevens F J, Solomon A, Schiffer M. BenceJones proteins: a powerful tool for the fundamental study of proteinchemistry and pathophysiology. Biochemistry. 1991; 30: 6803-5). In 1993Mei Sun et al., (Sun M, Li L, Gao Q S. Paul S. Antigen recognition by anantibody light chain. J Biol. Chem. 1994; 269: 734-8) reported that apurified L-chain from a monoclonal antibody against vasoactiveintestinal polypeptide (VIP) displayed sequence-specific and highaffinity binding to VIP. Accordingly, Nishimura E et al., (Nishimura E,Mochizuki K, Kato M, Hashizume S, Haruta H, Shirahata S, et al.Recombinant light chain of human monoclonal antibody HB4C5 as apotentially useful lung cancer-targeting vehicle. Hum Antibodies. 1999;9: 1 11-24) reported the recombinant production of a λ light chainexhibiting a significantly higher activity of binding to the antigen ascompared with the intact antibody. Furthermore, the authors presentedevidence that this recombinant light chain could serve as a potentiallyuseful vehicle for clinical use such as radioimmunoimaging andradioimmunotherapy of lung cancers. It has been also reported thatantibody L-chain dimers produced by a mouse hybridoma reacted to humanmelanoma tissues (Masat L, Wabl M Johnson J P. A simpler sort ofantibody. Proc Natl Acad Sci USA. 1994; 91: 893-6). In 1998 Pereira B etal, (Pereira B, Benedict C R, Le A, Shapiro S S, Thiagarajan P.Cardiolipin binding a light chain from lupus-prone mice. Biochemistry.1998; 37: 1430-7) showed that a single L-chain variable sequencecontains all the determinants necessary for cardiolipin binding, with anaffinity similar to that of the intact antibody. More recently,Dubnovitsky A P et al, (Dubnovitsky A P, Kravchuk Z I, Chumanevich A A,Cozzi A, Arosio P, Martsev S P. Expression, refolding, andferritin-binding activity of the isolated V_(L)-domain of monoclonalantibody Fl 1. Biochemistry (Mosc). 2000; 65: 1011-8) reported that therecombinant V_(L)-domain of a monoclonal antibody against ferritinpreserved its antigen binding function and that the antigen bindingconstant of the V_(L)-domain is comparable to that of the full-lengthparental antibody.

The results reported herein represent the first experimental evidence ofa L-chain dimer having a function-blocking activity. More specifically,the present work demonstrated that both the recombinant and chimeric 4C5consisting of an L-chain dimer have a high specific activity as examinedby immunoblotting and immunofluoresence experiments using breast cancercells. Furthermore and similarly to the paternal antibody they exhibitfunction-blocking properties as judged by the in vitro wound healingassay. These results provide a new understanding for comprehensiveapproaches in the clinical applications of L-chain antibodies.Recombinant L-chain antibodies have a number of advantages over usualantibodies and VH antibodies when intended for human therapeutics, i.e.,easy and reproducible production with constant quality, highersolubility and resistance to aggregation, faster clearance fromcirculation, among others. Especially in the case of mAb 4C5, twoadditional attributes make its chimerization valuable for therapeuticuse. First, its ability to functionally inhibit specifically the HSP90pool localized on the cell surface without interfering with HSP90functions inside the cell; and secondly, its minimal immunogenicity in aclinical setting due to its largely human composition. Therefore, thedevelopment of chimeric Ab 4C5 represents an important step in thedevelopment of novel HSP90 inhibitors for the treatment of humanmalignancies.

Example 8 Monoclonal Ab 4C5 Inhibits the Metastatic Deposition of BreastCancer Cells into the Lungs of SCID Mice_([PC9])

To explore the inhibitory effects of mAb 4C5 against the metastaticbehavior of malignant breast cancer cells in vivo, MDAMB453 breastcancer cells labeled with the fluorescent dye DiI were injectedintravenously in SCID mice, either in the presence or in the absence of100 μg/ml of mAb 4C5. Twenty four hours after the injection, mice wereeuthanized and the metastatic deposits of cells were traced andevaluated in the lungs of both the control and the mAb 4C5 treatedgroups.

FIG. 8 shows that mAb 4C5 inhibits the metastatic deposition of breastcancer cells into the lungs of SCID mice. At the macroscopic level animportant number of metastatic cell deposits (arrow in FIG. 8A) wasobserved in control animals as compared to mAb 4C5 treated mice. Thiswas confirmed microscopically where a very important decrease in thedeposition of cancer cells was detected in the mAb 4C5 treated mice(arrows in FIG. 8B). Quantification of the metastatic deposit formationrevealed a 86.67% inhibition in the mAb 4C5 treated mice when comparedwith control mice (FIG. 8C). In order to further visualize thedeposition of MDAMB453 cells in the lung tissue, we performed a 3Dreconstitution of a cryosection derived from a control animal where theinfiltration of metastatic deposits of MDAMB453 cells in the lung tissueis clearly demonstrated (arrow in FIG. 8D). It is of interest to notethat in the mAb 4C5 treated mice, MDAMB453 cells were often observedstagnating on the inner surface of large pulmonary blood vessels whereasno such images could be detected in the control animals (arrows in FIG.8E). Quantification of this occurrence revealed that in 58.76% of thepulmonary vessels visualized in the, mAb4C5 treated animals, MDAMB453cells were observed stagnating on the inner surface of the vessels. Incontrast, only 15.4% of the vessels observed in the control animalsshowed intravascular retention of the cancer cells (FIG. 8F) Theseresults demonstrate that mAb 4C5 inhibits the metastatic deposition ofMDAMB453 cancer cells into the lungs of SCID mice.

Example 9 Monoclonal Ab 4C5 Inhibits the Metastasis of Triple-negativeBreast Cancer Cells into the Lungs of SCID Mice

FIG. 9 shows that monoclonal antibody 4C5 inhibits metastasis ofMBDAMB231 triple-negative breast cancer cells in the lungs of SCID miceby 60%. In the experiment shown in FIG. 9, SCID female mice aged 6-8weeks were injected intravenously with 5×105 DiI-labeled MDAMB231 breastcancer cells. One day post-injection the mice were split in 2 groups; 5mice received 200 μg PBS buffer containing an irrelevant antibodyintraperitoneally, daily for 2 weeks (control group), while 5 micereceived 200 μg mAb 4C5 per mouse intraperitoneally, daily for 2 weeks(experimental group, see Methods). At the end of the experiment (day 72)lungs were obtained, counter-stained with Topro-3 and visualized byconfocal microscopy. Under these conditions mAb 4C5 inhibited MDAMB231metastasis by 38% (p<0.05).

Example 10 Monoclonal Ab 4C5 Delays Primary Tumor Growth of MDAMB231Triple Negative Breast Cancer Cells Growing Orthotopically in SCID Mice

To assess the effect of mAb 4C5 on orthotopic primary breast cancergrowth, female SCID mice aged 6-8 weeks were injected under the mammaryfat pad with 5×10⁶ MDAMB231 triple-negative breast cancer cells. One daypost-injection the mice were split in 2 groups; 5 mice received 200 μgPBS buffer containing an irrelevant antibody intraperitoneally, dailyfor 2 weeks (control group), while 6 mice received 200 μg mAb 4C5 permouse intraperitoneally, daily for 2 weeks (experimental group, seeMethods). Both groups were then left untreated until the end of theexperiment (day 40). The results from a representative experiment shownin FIG. 10 suggest that mAb 4C5 inhibits the primary tumor growth ofMDAMB231 cells by 65% (p=0.007).

The results described above were obtained using the following methodsand materials.

RNA and DNA Manipulations.

Total RNA was isolated from 10⁷ hybridoma cells, using absorption onsilica-based fiber matrix following the manufactures instructions(Absolutely RNA Mini Prep kit obtained from Stratagene). For Northernblot analysis, 25 ug RNA were separated by electrophoresis on agarosegel, transferred to a positively charged nylon membrane (Zeta-Probeobtained from Biorad) and hybridized with a labeled 1.2-kb heavy chainIgGla cDNA probe. For amplification of the V_(H) and V_(L) antibodyfragments, specific first strand cDNAs were synthesized by reversetranscription and using polyT oligonucleotide as primer, according tothe manufacturer's instructions (I^(S) Strand cDNA Synthesis kit forRT-PCR obtained from Roche). The cDNAs were subsequently amplified byPCR, using specific mouse immunoglobulin primers as shown in table 1.

TABLE 1 Murine IgG 1 Immunoglobulin PCR Primers(SEQ ID NOS 11-21, respectively, in order of appearance)Heavy-chain Fd 3′ primer H3: 5′-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3′ Heavy-chain variable 5′ primersH5′1: 5′-AGG TCC AGC TGC TCG AGT CTG G-3′H5′2: 5′-AGG TCC AGC TGC TCG AGT CAG G-3′H5′3: 5′-AGG TCC AGC TTC TCG AGT CTG G-3′H5′4: 5′-AGG TCC AGC TTC TCG AGT CAG G-3′H5′5: 5′-AGG TCC AAC TGC TCG AGT CTG G-3′H5′6: 5′-AGG TCC AAC TGC TCG AGT CAG G-3′H5′7: 5′-AGG TCC AAC TTC TCG AGT CTG G-3′H5′8: 5′-AGG TCC AAC TTC TCG AGT CAG G-3′ Murine κ light-chain 3′ primerL3′: 5′-GCG CCG TCT AGA ATT AAC ACT CAT TCC  TGT TGA A-3′Murine light-chain variable 5′ primersL5′6: 5′-CCA GAT GTG AGC TCG TCA TGA CCC AGT  CTC CA-3′

Amplified L-chain cDNA fragment was inserted into the SacI/XbaIrestriction sites of the pComb3HSS phagemid (generous gift of Drs. C. F.Barbas and D. R. Burton, The Scripps Research Institute, La Jolla,Calif.) and sequenced in both directions at the sequence facility of theInstitute of Molecular Biology and Biotechnology (IMBB). The germlinecounterpart of the rearranged V_(L) sequence was analyzed using theNational Center for Biotechnology Information IgBLAST server(http://www.ncbi.nlm.nih.gov/igblast) and the sequence was aligned usingClustalW software. Construction of mouse-human chimeric Ab 4C5

The mouse-human chimeric antibody was constructed by fusing the mAb 4C5V_(L) cDNA to a human C_(K) gene segment. Briefly, the κ light chain ofmAb 4C5 was subcloned into pBluescript SK-plasmid, and then theBsgI/XbaI fragment containing the mouse C_(K) region was replaced by theBsgI/XbaI restriction fragment containing the human C_(K). Finally, thechimeric κ L-chain gene of mAb 4C5 was inserted into the SacI/XbaI sitesof pComb3HSS. All DNA manipulations were performed according to(Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989).

Expression and Purification of the Mouse and Chimeric L-chains

Soluble recombinant antibody light chains were produced from individualbacterial colonies as described previously (Barbas, C. F. 3 and Burton,D. R., Monoclonal Antibodies from Combinatorial Libraries, 1994) and thebacterial periplasmic fractions were extracted according to (Charlton,K. A., Expression and Isolation of Recombinant Antibody Fragments in E.coli., Methods Mol. Biol. 2004; 248:245-54) and purified by affinitychromatography using a Protein L column (PIERCE, USA) on an FPLC AKTAsystem (Amersham Biosciences, Piscataway, N.J.), according to themanufacture's instructions.

Reducing and Non Reducing SDS-PAGE

Antibody-containing FPLC fractions were pooled together and concentratedby centrifugation through a dialysis membrane (Amicon Ultra centrifugalfilter devices obtained from Millipore). The electrophoretic motilitiesof the mouse, recombinant, and chimeric antibodies were tested underreducing and non-reducing conditions using the Laemmli discontinuoussystem as described before (Laemmli, U.K., Cleavage of StructuralProteins During the Assembly of the Head of Bacteriophage T4., Nature.1970; 227: 680-5). Antibodies were visualized either by Coomasie-R stainor by western blot using specific secondary HRP-conjugated anti-kappachain antibodies.

Preparation of cell lysates, immunoprecipitation and western blotanalysis MDAMB453 cancer cell lysates were obtained and quantified aspreviously described (Thomaidou, D. and Patsavoudi, E., Identificationof a Novel Neuron-specific Surface Antigen in the Developing NervousSystem, by Monoclonal Antibody 4C5., Neuroscience. 1993; 53: 813-27),and equal amounts of total protein were subjected to SDS-PAGE andtransferred onto nitrocellulose. The membranes were blotted for 40 minat room temperature with non-fat dry milk (5%) in tris-buffered saline(“TBS”) containing 0.05% Tween-20, to block non-specific binding sitesand were then incubated with the specific primary antibodies overnightat 4° C. The membranes were washed with 0.3% bovine serum albumin(“BSA”) in TBS and incubated with horseradish peroxidase-labeledanti-kappa chain secondary antibodies for 2 hours at room temperature.After washing with TBS, the bound antibody complexes were detected usingDAB or/and a chemiluminescence reagent (ECL chemiluminescence reagentobtained from Amersham) and exposure to X-ray film (XOMAT-AR filmobtained from Kodak) as described by the manufacturers.

Immunoprecipitation was performed as previously described (Sidera, K. etal., A Critical role for HSP-90 in Cancer Cell Invasion InvolvesInteraction with the Extracellular Domain of HER-2., J. Biol. Chem.,2008; 283: 2031-41). In brief, equal amounts of pre-cleared MDAMB453cancer cell lysates were incubated with antibodies overnight at 4° C.The immunocomplexes were then incubated for 2 hours at room temperaturewith protein G-sepharose, and washed 3 times with lysis buffer. Boundproteins were analyzed by gel electrophoresis followed by western blot.For all immunoprecipitation experiments, negative controls wereperformed using irrelevant IgGs.

Cell Cultures and Immunofluorescence

MDAMB453 breast cancer cell line was maintained in RPMI supplementedwith 10% fetal bovine serum (“FBS”). For immunofluorescence studies,cells were plated on poly-Llysine coated coverslips, at a density of5×104 cells/well in a 48-well plate and cultured in RPMI mediumsupplemented with 10% FBS. After 24 h, cells were fixed and processedfor indirect immunoflorescence, as previously described (Thomaidou, D.and Patsavoudi, E., Identification of a Novel Neuron-specific SurfaceAntigen in the Developing Nervous System, by Monoclonal Antibody 4C5.,Neuroscience. 1993; 53: 813-27). Live MDAMB453 cells were labeled byindirect immunofluorescence as previously reported (Sidera, K. et al,Involvement of Cell Surface HSP90 in Cell Migration Reveals a Novel Rolein the Developing Nervous System., J. Biol. Chem., 2004; 279: 45379-88).Alexa546-labeled phalloidin (Molecular Probes, Eugene, Oreg.) was usedto visualize F-actin. For all experiments, controls were performed byomitting the primary antibodies or/and by using an IgG2a monoclonalantibody against the unrelated neuronal protein BM88. Immunofluorescencewas analyzed by confocal microscopy using a Leica TSC confocalmicroscope.

Antibody Internalization Assay

MDAMB453 cells were incubated while in culture with the antibodies atequal concentrations for 2, 8 and 24 h. The cells were then washed inRPMI and fixed. For detection of any internalized antibody, cells werepermeabilized with 0.1% Triton X-100 in PBS and subsequently incubatedwith Alexa488-conjugated secondary antibody (Molecular Probes, Eugene,Oreg.). For all experiments, controls were performed as mentioned above.

Wound Healing Assay

The assay was performed as previously described (Sidera, K. et al., ACritical role for HSP-90 in Cancer Cell Invasion Involves Interactionwith the Extracellular Domain of HER-2., J. Biol. Chem., 2008; 283:2031-41). Briefly, MDAMB453 and B16 F 10 were plated on poly-L-lysinecoated cover slips in a 48-well plate at a density of 1×10⁵ and 2.5×10⁵cells/well, respectively. After 24 hours the medium was changed to serumfree RPMI and 16 hours later, a cell free area was generated by gentlyscratching the cell monolayer with a sterile yellow Gil son-pipette tip,thus resulting in the formation of an approximately 1 mm-wide cell-freearea. Immediately after scratching, the medium was replaced with freshmedium, containing anti-HSP90α antibody, mAb 4C5, recombinant 4C5, orchimeric 4C5. All agents were maintained in the culture for the durationof the assay.

Control cultures were grown either in culture medium alone (RPMI) or inculture medium containing an IgG2a monoclonal antibody (concentratedserum free hybridoma supernatant), against the unrelated protein BM88.Migration of cancer cells within the gap was monitored microscopicallyat given time intervals, using a Leica DM IL inverted microscope,equipped with a LEICA DM300 video camera connected to a computer.Migration distance was estimated by acquiring and analyzing digitalimages, using the Image Pro Plus analysis software and expressed as thepercent distance covered by cells in control cultures. Statisticalanalysis was performed by using the Student's T-test.

Trypan Blue Staining.

At the end point of the wound-healing assay, MDAMB453 cells wereincubated for 5 min with 0.4% trypan blue in PBS. Excess staining wasremoved and cells were visualized in a Leica microscope.

Clonogenic Assay

Clonogenic assay was performed as previous described (Franken N et al.,Clonogenic assay of cells in vitro Nature Protocols 2006; 1; 2315-2319).Briefly, MDAMB231 single cell suspensions were seeded on 6-well platesat a density of 500 cells/well and cultured either in culture mediumalone or in culture medium containing 200 μg/ml chimeric 4C5. Media werereplaced every 3 days and colonies were left to grow for 2 weeks beforestaining with Giemsa.

Assay of Breast Cancer Cell Metastatic Deposit and Metastasis in LungTissue

SCID mice originally purchased from Jackson Laboratory or Harlan Labswere bred and maintained under specific pathogen free conditions at theExperimental Animal Unit of the Hellenic Pasteur Institute. All of theexperiments with animals were done in accordance with the guidelinesapproved by the Ethical Committee of the Hellenic Pasteur Institute. Thein vivo metastatic deposit formation assay was performed as follows.Briefly, cultured MDAMB453 or MDAMB231 cells were pre-incubated with DiIfor 1 hour, washed twice with PBS, trypsinized and made up to the celldensity of 10⁶/300 μL in PBS in the absence or presence of 100 μg/ml ofan irrelevant antibody BM88 (Patsavoudi E, Hurel C, Matsas R:Purification and characterization of neuron-specific surface antigendefined by monoclonal antibody BM88. J Neurochem 1991, 56:782-8) or 100μg/ml of mAb 4C5. Twenty 8-10-week-old female SCID mice were injectedthrough the tail vein with 0.3 ml of the above cell preparations. Theanimals were divided into two equal groups: the control group injectedwith cells dialysed in PBS or the irrelevant antibody and the mAb 4C5treated group. The animals were euthanized 24 hours later (FIG.8-metastatic deposits) or 72 days later (FIG. 9, lung metastasis assay).(In the metastatic deposit assays, a peristaltic pump was additionallyconnected to the left ventricle of the heart was employed to wash outthe remaining blood from mouse lungs, by pumping 200 ml of salinebuffer. This procedure ensures that all cancer cells which are notattached, either on the inner surface of the blood vessels or on thelung tissue, are removed.) Finally the lungs were perfused with 4%formalin solution and then embedded in OCT solution in order to performcryosections. Each lung was sectioned with the cryotome and each sectionwas counter stained with Dapi and visualised with a confocal microscope.In ten randomly chosen slides covering the whole of the lung tissue theMDAMB453 cells were calculated. The same experiment was performed twicewith similar results.

Orthotopic Breast Cancer Mouse Xenograft Studies

The orthotopic breast cancer xenograft tumor mouse model was performedas previously described (Wei Yan et al The journal of BiologicalChemistry vol 285 No. 18, pp 14042-14051, Apr. 30, 2010). Briefly,cultured MDAMB231 cells were pre-incubated with DiI for 1 hour, washedtwice with PBS, collected with cell scraper and made up to the celldensity of 5×10⁶/100 μL in PBS free of calcium and magnesium. Six toeight week-old female Balb-c SCID mice were injected under the mammaryfat pad with 5×10⁶ DiI stained MDAMB231 cells and subsequently dividedinto 2 groups, experimental and control. One day after injectionexperimental mice received 200 μg mAb 4C5 (1 μg/μl) i.p. daily for 2weeks whereas control mice received the same volume of vehicle (PBS).Tumor growth rate was monitored at weekly intervals and calculated basedon the equation, tumor volume (mm³)=length×width²×0.52. At the end ofthe experiment animals were sacrificed and mouse tumor samples andsamples from the lung and the liver were processed forimmunohistochemistry.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

Incorporation by Reference

The entire contents of all patents published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

SEQUENCE LISTING The Sequence Listing, SEQ ID NO: 1, corresponds to the nucleotide sequence encoding mAb 4C5.GAGCTCGTCATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAACAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCTCGGCTCACGTTCGGTGCTGGGACCAGGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCG

GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTThe Sequence Listing, SEQ ID NO: 2, corresponds to the amino acid sequence of mAb 4C5.ELVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTINSLEYEDMGIYYCLQYDEFPRLTFGAGTRLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNECThe Sequence Listing, SEQ ID NO: 3, corresponds to the nucleotide sequence of chimeric 4C5.GAGCTCGTCATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAACAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCTCGGCTCACGTTCGGTGCTGGGACCAGGCTGGAGCTGAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGGACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTThe Sequence Listing, SEQ ID NO: 4, corresponds to the amino acid sequence of chimeric 4C5 and of ch4C5ΔCys (the latter however lacks the undelined C-terminal cysteine at the 3′ end)ELVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTINSLEYEDMGIYYCLQYDEFPRLTFGAGTRLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECThe Sequence Listing, SEQ ID NO: 5, corresponds to the amino acid sequence of CDR1 of 4C5: KASQDINSYLSThe Sequence Listing, SEQ ID NO: 6, corresponds to the amino acid sequence of CDR2 of 4C5: RANRLVDThe Sequence Listing, SEQ ID NO: 7, corresponds to the amino acid sequence of CDR3 of 4C5: LQYDEFPRLTThe Sequence Listing, SEQ ID NO: 9, corresponds to the nucleotide sequence of chimeric 4C5 for improved expression in bacterial cells.GAGCTCGTCATGACCCAGAGCCCGAGCAGCATGTATGCAAGCCTGGGCGAACGTGTGACCATCACCTGCAAAGCGAGCCAGGATATTAATAGCTATCTGTCTTGGTTTCAGCAGAAACCGGGCAAAAGCCCGAAAACCCTGATTTATCGTGCAAACCGTCTGGTAGATGGCGTGCCGTCACGTTTTAGCGGTTCTGGCAGCGGCCAGGATTATAGCCTGACCATTAACAGCCTGGAATATGAAGATATGGGCATTTATTATTGCCTGCAGTATGATGAATTTCCGCGTCTGACGTTTGGCGCGGGCACCCGTCTGGAACTGAAACGTACCGTTGCGGCACCGAGCGTGTTTATTTTTCCGCCGAGCGATGAACAGCTGAAAAGTGGCACCGCGAGCGTTGTGTGCCTGCTGAATAACTTTTATCCGCGTGAAGCCAAAGTACAGTGGAAAGTGGATAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGTCTAGCACCCTGACGCTGAGCAAAGCAGATTATGAAAAACATAAAGTGTATGCCTGCGAAGTGACCCATCAG

The Sequence Listing, SEQ ID NO: 10, corresponds to the nucleotide sequence of chimeric 4C5 for improved expression in bacterial cells.GAGCTCGTCATGACCCAGAGCCCGAGCAGCATGTATGCAAGCCTGGGCGAACGTGTGACCATCACCTGCAAAGCGAGCCAGGATATTAATAGCTATCTGTCTTGGTTTCAGCAGAAACCGGGCAAAAGCCCGAAAACCCTGATTTATCGTGCAAACCGTCTGGTAGATGGCGTGCCGTCACGTTTTAGCGGTTCTGGCAGCGGCCAGGATTATAGCCTGACCATTAACAGCCTGGAATATGAAGATATGGGCATTTATTATTGCCTGCAGTATGATGAATTTCCGCGTCTGACGTTTGGCGCGGGCACCCGTCTGGAACTGAAACGTACCGTTGCGGCACCGAGCGTGTTTATTTTTCCGCCGAGCGATGAACAGCTGAAAAGTGGCACCGCGAGCGTTGTGTGCCTGCTGAATAACTTTTATCCGCGTGAAGCCAAAGTACAGTGGAAAGTGGATAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGTCTAGCACCCTGACGCTGAGCAAAGCAGATTATGAAAAACATAAAGTGTATGCCTGCGAAGTGACCCATCAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTTAATCGTGGCGAA

What is claimed is:
 1. An isolated chimeric antibody comprising thesequence of SEQ ID NO: 4, wherein the chimeric antibody has a murinekappa L-chain that lacks heavy chains, wherein the murine immunoglobulinκ light chain constant domain (C_(K)) is replaced with the correspondinghuman C_(K) domain or a fragment thereof, and wherein the antibodyspecifically binds HSP90 and is capable of reducing the growth and/orthe invasiveness of a neoplastic cell.
 2. The chimeric antibody of claim1, wherein the antibody is a humanized antibody or is isolated from aculture of prokaryotic or eukaryotic cells.
 3. The chimeric antibody ofclaim 1, wherein the antibody is a monomer, a dimer or a multimer. 4.The chimeric antibody of claim 1, wherein the chimeric antibody has areduced capacity to induce an immune response in a human subject,relative to a conventional murine antibody.
 5. The chimeric antibody ofclaim 1, wherein the ability of the antibody or a fragment thereof toreduce growth and invasiveness is assayed using a cancer cell clonogenicassay, a wound healing assay, a lung metastatic deposit formation assay,a lung metastasis inhibition assay, a breast cancer primary tumor growthinhibition assay, by detecting actin rearrangement, by detectinglamellipodia development, or by detecting another morphological markerof invasiveness, by detecting inhibition of metastatic lung deposits, bydetecting inhibition of lung metastasis, by detecting delay of primarygrowth tumors implanted orthotopically in mouse fat pads, or bydetecting another marker of efficacy, respectively.
 6. An isolatedpolypeptide comprising the sequence of SEQ ID NO:
 4. 7. An isolatedpolynucleotide encoding the chimeric antibody of claim
 1. 8. An isolatedpolynucleotide encoding the isolated polypeptide of claim
 6. 9. Anexpression vector comprising the polynucleotide of claim 7 positionedfor expression in a cell.
 10. A cell comprising the expression vector ofclaim
 9. 11. The cell of claim 10, wherein the cell is a prokaryotic oreukaryotic cell.
 12. A method for producing a chimeric antibody of claim1, the method comprising culturing the cell of claim 10 under conditionssuitable for expression of the chimeric antibody, and isolating thechimeric antibody from the cultured cell.
 13. A pharmaceuticalcomposition for the treatment of neoplasia comprising a therapeuticallyeffective amount of the chimeric antibody of claim
 1. 14. Apharmaceutical composition for the treatment of neoplasia comprising atherapeutically effective amount of the isolated polypeptide of claim 6.15. A kit for the treatment of a neoplasia, the kit comprising atherapeutically effective amount of the chimeric antibody of claim 1.16. An isolated chimeric antibody comprising an amino acid sequencehaving at least 90% or 95% identity to SEQ ID NO: 4, wherein thechimeric antibody has a murine kappa L-chain, wherein the murineimmunoglobulin κ light chain constant domain (C_(K)) is replaced withthe corresponding human C_(K) domain or a fragment thereof, wherein theantibody specifically binds HSP90 and is capable of reducing the growthand/or the invasiveness of a neoplastic cell, and wherein the antibodycomprises the complimentarity determining regions of SEQ ID NO: 5, SEQID NO: 6, and SEQ ID NO:
 7. 17. The isolated chimeric antibody of claim16, wherein the amino acid sequence has at least 95% identity to thesequence of SEQ ID NO:
 4. 18. The chimeric antibody of claim 16, whereinthe antibody is a monomer, a dimer or a multimer.
 19. An isolatedpolynucleotide encoding the chimeric antibody of claim
 16. 20. Anexpression vector comprising the polynucleotide of claim 19 positionedfor expression in a cell.
 21. A cell comprising the expression vector ofclaim
 20. 22. The cell of claim 21, wherein the cell is a prokaryotic oreukaryotic cell.